An in vitro system was used to study DNA synthesis in lysates of Escherichia coli cells which had been grown in the presence of ethionine. Such lysates showed a reduced capacity to incorporate (3H)TTP into high-molecular-weight material. Activity could be restored by incubation with S-adenosyl methionine and ATP. S-adenosyl methionine-reactivated TTP incorporation required the presence of DNA polymerase I, ATP, and all four deoxyribonucleotide triphosphates. DNA polymerase III was not required. Methylation dependent replication in vitro and in vivo in the presence of chloramphenicol. The synthesis product appeared initially as small pieces which could join in vivo even in the absence of ligase. A conditional lethal mutation, dna298, has been isolated. It is linked to serB. The mutant doubles its DNA content at 42 degrees and then DNA synthesis ceases. Replication can be resumed if the temperature is lowered but not in Ethionine. Some evidence indicates that the mutant may regulate the normal restriction-modification system. This work will be continued.